1. Field of the Invention
This invention relates to the field of phthalocyanin compounds and their use as biological stains or as dyes.
2. Brief Description of the Prior Art
The differentiation of tissue and cell types by their staining properties has long been a major tool in histology and hematology. Several phthalocyanin compounds are known for use as such. The first of these to be discovered is called Alcian Blue and has the following structure: ##STR2##
Alcian Blue has been used for the differential staining of basophils. The copper phthalocyanin cationic dyes are not sufficiently specific to achieve the selective staining of basophils when used alone because they also stain other cells which possess polynucleotides, e.g., DNA and RNA. In addition, basophils stain because of the unique presence in them of heparin, a sulfated polysaccharide. One way of establishing the desired selectivity is to combine it with lanthanum chloride which masks the polynucleotide phosphate groups and thereby prevents them from binding the phthalocyanin anion.
The use of Alcian Blue requires a closely controlled, highly acidic pH and it is heat labile. At alkaline pH and when exposed to heat, Alcian Blue forms particulates (insoluble dyes). This tendency to precipitate has been a longstanding problem in Alcian Blue-containing reagents. Automated analysis instruments contain components such as filters which collect these precipitates. This can interfere with the reliability of the determinations being made and even the operation of instruments on which this method is performed. It has nonetheless been considered the dye of choice because of its specificity and distinct color. For more background information on Alcian Blue, see Gilbert, et al, Basophil Counting With A New Staining Method Using Alcian Bue, Blood, 46: 279-286 (1975).
Other phthalocyanin dyes have since been developed. For example, Bloom, et al, Histochemie, 2: 48-57 (1960) shows the use of underivatized Astra Blue (free base) to stain biological tissues containing mucopolysaccharides, particularly mast cells. The Astra Blue free base is used at pH 0.2-0.3, which gives it a positive charge. The low pH allows selectivity because of the inherent strength of sulfuric acid derivatives, e.g., heparin, which is ionized at pH 0.3, as compared to the weakness of phosphoric acid derivatives, e.g., DNA, which is not ionized at pH 3.0.
Inagaki, Acta Hematologica Japonica, 32(4): 642-647 (1969), describes for staining basophil and mast cell granules using free base Astra Blue and a fixative solution of Acridine Orange in methanol (pH about 0.3). Inagaki examined saturated cetyl pyridinium chloride in absolute methanol and saturated Acridine in absolute methanol for the fixation of peripheral blood and bone marrow smears. Cetyl pyridinium chloride securely preserved the basophil granules and the mast cell granules, but the Astra Blue staining tended to be prevented. Acridine could not preserve these cell granules sufficiently in the above described procedure.
Scott, The Molecular Biology Of Histochemical Staining By Cationic Phthalocyanin Dyes; The Design of Replacements for Alcian Blue, J. Microscopy, 119: 373-381 (1979) indicates that the commercially available Astra Blue free base (Bayer AG, Leverkusen, W. Ger.), shown as IIa below, can be methylated by dimethylsulfate to yield the quaternized (methylated Astra Blue, shown as IIb. ##STR3##
Although no use of these compounds in staining cellular or tissue materials was demonstrated by Scott, the desire to find compounds other than Alcian Blue for this purpose was expressed. This trimethyl quaternary salt and other phthalocyanin dyes were shown to stain polyanions such as heparin, polygalacturonate DNA and RNA. Thus, Scott teaches that these compounds do not selectively react with heparin as is required for differentiation of basophils.
In summary, Alcian Blue and Astra Blue free base have been the only compounds of this type which have been known to differentiate basophils from other white blood cells. The instability of Alcian Blue reagent has been a longstanding problem which has not been overcome. Thus, there remains the need for compounds which selectively stain basophils, in contrast to other white blood cells, and which are stable over time in solution.